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Fast and Automatic Mapping of Disulfide Bonds in a Monoclonal Antibody using SYNAPT G2 HDMS and BiopharmaLynx 1.3Hongwei Xie and Weibin ChenWaters Corporation, Milford, MA, U.S.
IN T RO DU C T IO N
Disulfide bond formation is critical for establishing and maintaining proper
three-dimensional folding and therefore functions of therapeutic proteins, e.g.,
monoclonal antibodies (mAbs). Localization and assignment of disulfide bonds
are an important aspect of protein structural analysis.
However, the identification of disulfide pairing in a protein with multiple
cysteine residues is generally more time-consuming and challenging than the
determination of the protein sequence due to incomplete disulfide bond formation
and disulfide bond scrambling.1,2
The combination of enzymatic digestion with liquid chromatography-mass
spectrometry (LC/MS) is frequently applied as a routine method for the
assignment of disulfide bonds.3,4 However, peptide mapping via an LC/MS
approach is traditionally labor-intensive and time-consuming in data processing
and interpretation.
Furthermore, the assignment of disulfide linkages sometime is inconclusive when
a protein contains multiple cysteines due to the significantly increased number
of possible disulfide-bonded peptide isomers.1 For example, there are totally
105 possible disulfide-bond pairing schemes for a protein containing eight
cysteine residues (while an IgG1 mAb typically has 32 or more cysteines in its
two light chains and two heavy chains).
This, plus the reality of potential disulfide bond scrambling, makes the disulfide
bond assignment extremely difficult when the analysis is performed manually.
An automated workflow is highly preferred for quick assessment of
heterogeneities of therapeutic proteins caused by the disulfide linkages.
Recently, we have developed an integrated peptide mapping workflow for protein
sequence confirmation and characterization using a UPLC®/MSE method for data
collection and a targeted bioinformatic tool, the BiopharmaLynx™ Application
Manager for MassLynx™ Software, for automated data processing and annotation.5
The data independent MSE approach alternatively collects mass spectrometric
data of precursors and fragments of eluting peptides from protein enzymatic
(e.g., tryptic) digests in an unbiased manner.6 The integrated workflow overcomes
most shortcomings of traditional peptide mapping methods7 and has been
demonstrated to be fast, robust and suitable for routine analysis of recombinant
WaT e R s sO lU T IO Ns
ACQUITY UPLC
SYNAPT G2 HDMS
BiopharmaLynx Application Manager
MassLynx Software
k e y W O R D s
Disulfide bonds, peptide map
a P P l I C aT IO N B e N e F I T s
In this study, an advanced LC/MSE peptide
mapping workflow is demonstrated for the
detection and identification of disulfide linkages,
including scrambled disulfide linkages, in a
recombinant IgG1 mAb. We develop a viable,
integrated approach for automatic mapping or
monitoring of disulfide bonded peptides in a
protein with multiple cysteines.
2Fast and Automatic Mapping of Disulfide Bonds in a Monoclonal Antibody using SYNAPT G2 HDMS and BiopharmaLynx 1.3
e X P e R IM e N Ta l
LC conditionsLC system: ACQUITY UPLC
Column: ACQUITY UPLC BEH300 C4, 1.7 µm, 2.1 x 150 mm (p/n 186004487) or
ACQUITY UPLC BEH300 C18, 1.7 µm, 2.1 x 150 mm (p/n 186003687)
Column temp.: 65 °C
Flow rate: 200 µL/min
Injection vol.: 5 µL (partial loop injection mode)
Mobile phase A: 0.1% FA in water
Mobile phase B: 0.1% FA in ACN
Gradient: 1–40% in 90 min
MS conditionsMS system: SYNAPT HDMS G2
Ionization mode: ESI+
Acquisition mode: MSE
Low collision energy: 4 eV
Elevated collision energy: Ramping from 22 to 48 eV
Capillary voltage: 3.0 kV
Cone voltage: 30 V
Scan mode: Resolution mode (resolution ≥ 20000)
Scan time: 0.5 second
Source temp.: 100 °C
Desolvation temp.: 350 °C
Mass range: 100 to 2500 Da
Mass accuracy: Lockmassed by GFP sprayed from lockmass channel every 30 seconds
Data managementBiopharmaLynx 1.3 Application Manager
for MassLynx Software
Materials and reagentsEndoproteinase Lys-C (Wako) and iodoacetamide (Sigma)were used along with RapiGest (Waters, p/n 186001861).The IgG1 mAb Trastuzumab digests were prepared byincubating the sample (protein/Lys-C of 50:1) in 50 mM trisbuffer (pH ~7.5) at 37 °C and the presence of 0.1% RapiGestSF for 18 hours. Prior to digestion, the protein was denaturedat 80 °C for 10 min. For testing artificial scramblingpotentially introduced during digestion, a digest preparedwithout alkylation was directly compared with a digestprepared in parallel with IAM alkylation before the digestion.
proteins such as therapeutic mAbs5 and subunit vaccines.8 The approach has also
been successfully applied to identify expected disulfide bonds in an IgG1 mAb.9
In this study, an advanced UPLC/MSE peptide mapping workflow is demonstrated
for the detection and identification of disulfide linkages (including scrambled
disulfide linkages) in a recombinant IgG1 mAb. The goal is to develop a viable
approach for automatic mapping or monitoring of disulfide bonded peptides in a
protein with multiple cysteines.
The significances of the integrated workflow include:
Enhanced mass resolution and mass accuracy using the SYNAPT■■ ® HDMS™
G2 QTof MS System,10 which is critical for acquiring high-quality MSE data for
large disulfide-bond-linked peptides with high molecular weight.
Automatic assignment of disulfide linkages by an upgraded BiopharmaLynx ■■
(version 1.3) in a randomized mode, which is important for the identification
of potential scrambled disulfide bonds in an automatic mode.
To reduce the complexity of protein digest mixture, the mAb was digested with
endoproteinase Lys-C. The obtained digests were separated by reversed-phase
LC with an ACQUITY UPLC® System, followed by online MSE detection and
BiopharmaLynx 1.3 analysis. Digests prepared with and without alkylation
protection of free sulfhydryl groups2 in the sample were used to differentiate
native scrambled disulfide linkages in the mAb from those potentially formed
during sample preparation stage.
R e sU lT s a N D D Is C U s s IO N
Like all IgG1 mAbs, the Trastuzumab antibody should have nine unique disulfide
bonds when formed properly. In-silico digestion (by Lys-Cp) shows that eight
expected disulfide linked peptides (or unique linkages) are generated after
digestion, including two in each of light chain, four in each of heavy chain, one
between light chain and heavy chain, and one between the two heavy chains
(which has two disulfide bonds connecting with).
To demonstrate that the workflow works properly to identify the formed disulfide
linkages, the collected LC/MSE data were first searched against the mAb
sequences with properly linked cysteines to form the 16 expected disulfide
bonds in the BiopharmaLynx method setting.
3Fast and Automatic Mapping of Disulfide Bonds in a Monoclonal Antibody using SYNAPT G2 HDMS and BiopharmaLynx 1.3
Figure 1. Identification procedure of disulfide linkage 1:K1–1:K4. A) UPLC/MS chromatograms showing the elution time of the linkage; B) MS spectrum; C) BiopharmaLynx interpretation and annotation; D) MSE spectrum.
Figure 1 shows the determination of the largest disulfide linkages 1:K1–1:K4 (an intra disulfide linkage of
light chain, monoisotopic mass 10657.12 Da). The disulfide-bonded peptide was detected and annotated based
on the precursor mass MH+ (Figure 1C) that was obtained from multiply-charged, isotope resolved raw MS
data (Figure 1B). The assignment was also confirmed by elevated-energy fragmentation MSE spectrum (also
charge-reduced, isotope-deconvoluted to singly-charged ions, as shown in Figure 1D) upon the retention time-
alignment (as highlighted in Figure 1A). The MSE spectrum not only contains b/y ions from the two individual
peptides (1:K1 and 1:K4), but also has ions corresponding to disulfide-bonded fragments from both peptides
such as 7019.27 Da (1/y39–2/y24) and 10431.19 Da (1/y39–2/y56), providing unequivocal evidences for
the disulfide linkage.
It should be stressed that the improved MS instrument resolution (see the inset in Figure 1B) is important to
obtain the correct monoisotopic masses for the precursors as well as fragments of the large peptides. A well-
resolved isotope pattern for such large peptides greatly facilitates the informatics tool for the identification of
large disulfide linkages such as 1:K1–1:K4.
Similarly, BiopharmaLynx identifies other expected disulfide linkages. The mass accuracy of all identified
disulfide bonded peptides was within 5.0 ppm. In addition, BiopharmaLynx is also capable of identifying the
disulfide-bonded peptides with one of the linked peptides containing miscleavages.
4Fast and Automatic Mapping of Disulfide Bonds in a Monoclonal Antibody using SYNAPT G2 HDMS and BiopharmaLynx 1.3
Figure 2. Identification and confirmation of disulfide linkages 2:K7–2:K8 and 2:K7–2:K8-9. A) BiopharmaLynx interpretation and annotation; B) MSE spectrum of 2:K7-2:k8; C) MSE spectrum of 2:K7–2:K8-9.
For example, both disulfide linkages 2:K7–2:K8 (without miscleavage) and 2:K7–2:K8-9 (with one
miscleavage) were identified (see corresponding peaks B and C in Figure 2A). Their MSE spectra are plotted
in Figures 2B and 2C, respectively, showing confident identification. This capability is especially important
because proteases such as Lys-C or trypsin tend to miss the cleavage site when a lysine (K) is followed by a
proline (P) residue.
It is observed that high percentage of organic solvent (10% or more acetonitrile) is needed in the sample to
help maintain the solubility of large disulfide-bonded peptides during LC/MS analysis. One disadvantage of
the use of high organic in sample buffer is that this could result in the elution of the smallest disulfide-bonded
peptides 1:K14–2:K13 (SFNRGEC=SCDK) in void volume.
5Fast and Automatic Mapping of Disulfide Bonds in a Monoclonal Antibody using SYNAPT G2 HDMS and BiopharmaLynx 1.3
Figure 3. A screen capture showing the Scrambled Disulfides function in BiopharmaLynx 1.3 data processing method setting.
One challenge in disulfide bond mapping is to identify those peptides formed unexpectedly, e.g., due to
scrambling. Therefore, the processing method was next created for Scrambled Disulfides (see the highlight
part in Figure 3), which allows for detection and identification of all possible disulfide pairing formed between
any two cysteines in the mAb. Lys-C digests of Trastuzumab with or without alkylation prior to digestion were
analyzed for this investigation.
6Fast and Automatic Mapping of Disulfide Bonds in a Monoclonal Antibody using SYNAPT G2 HDMS and BiopharmaLynx 1.3
Figure 4. Examples of identified scrambled disulfide linkages with automatic interpretation and annotation. A) BiopharmaLynx interpretation and annotation; B) MSE spectrum of scrambled disulfide linkage 1:K13–2:K16.
Using this new method, not only the expected disulfide linkages were identified, some minor scrambled
disulfide linkages were also detected. Figure 4A illustrates three identified scrambled disulfide linkages
(1:K13–2:K16, 1:K7–2:K21, 1:K14–2:K16) in the digest without alkylation. Again, elevated-energy
fragmentation (MSE) data were available for confirmation of the assignments (the MSE spectrum of
1:K13–2:K16 is plotted in Figure 4B as an example).
The scrambled 1:K13–2:K16 and 1:K14–2:K16 were only identified from the digest without alkylation and
not detected in the digest with alkylation, suggesting these were artifacts introduced by sample preparation.
The scrambled disulfide linkage 1:K7–2:K21 was identified in both digests (with or without alkylation),
however, its abundance is much lower in the alkylated sample compared to the non-alkylated one (see the
inset in Figure 4A). As expected, all the identified scrambled disulfide linkages are minor compared to the
expected disulfide linkages (as illustrated in Figure 4A).
The successful determination of both expected and scrambled disulfide linkages demonstrated that the
integrated workflow is capable of automatically mapping disulfide linkages in a protein. The enhanced function
of Scrambled Disulfides requires no manual efforts, and achieves completely automatic data processing for the
challenging task of disulfide bond mapping.
Waters Corporation 34 Maple Street Milford, MA 01757 U.S.A. T: 1 508 478 2000 F: 1 508 872 1990 www.waters.com
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CO N C lU s IO N
An integrated workflow, combining high mass resolution
UPLC/MSE with BiopharmaLynx 1.3, was developed for rapid
mapping of disulfide linkages in a recombinant IgG1 mAb. The
workflow was demonstrated to be capable of simultaneous
identification of both expected and scrambled disulfide linked
peptides. Assignment of disulfide bond linked peptides is
automated, based on accurate MS measurement and confirmed
by elevated collision energy MSE fragmentation data.
The improved mass resolution – 20,000 for resolution mode
and 40,000 for high-resolution mode – offered by the SYNAPT
G2 HDMS System greatly enhances the MS data quality for large
disulfide-linked peptides in enzymatic (e.g., Lys-C) digests. The
added Scrambled Disulfides function setting of BiopharmaLynx
1.3 makes it possible for identification of any potential disulfide
linkages of a protein in an automated mode.
This integrated approach should be applicable for routine mapping
and monitoring of disulfide linkages in mAbs and other therapeutic
proteins with multiple disulfide linkages.
Waters, ACQUITY UPLC, UPLC, and SYNAPT are registered trademarks of Waters Corporation. HDMS, BiopharmaLynx, MassLynx, and T he Science of What’s Possible are trademarks of Waters Corporation. All other trademarks are the property of their respective owners.
©2011 Waters Corporation. Produced in the U.S.A.June 2011 720003939EN AG-PDF